Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181

Abstract Background The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. Objectives To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019–20, and to evaluate the functional advantage of carrying both genes as opposed to only one. Methods The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different β-lactam antibiotics. Results WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. Conclusions The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.


Introduction
There has been a global emergence of carbapenem-resistant Enterobacterales with escalating resistance to all available β-lactam antibiotics, which WHO has highlighted as critical priority pathogens. 1The major mechanism underlying carbapenem resistance is the degradation of carbapenems via bacterial enzymes called carbapenemases.One such enzyme is NDM, which belongs to the Ambler class B β-lactamases and is encoded by bla NDM , which confers resistance not only to carbapenems but also to almost all β-lactams. 24][5] The OXA-48 enzyme is also a carbapenemase of concern.It is difficult to detect because of the low-level in vitro resistance to carbapenem antibiotics it confers, posing a challenge to clinical laboratories. 6,7Genes encoding OXA-48-like enzymes, such as bla OXA-48 , bla OXA-181 and bla OXA-232 , are globally widespread. 6,7The bla NDM and bla OXA-48 -like genes were identified as the second and third most prevalent carbapenemase genes among carbapenemaseproducing Enterobacterales (CPE), respectively, in a global surveillance programme conducted between 2008 and 2014. 8pecifically, bla OXA-181 and bla NDM-5 were the first and second most prevalent carbapenemase genes, respectively, among global carbapenemase-producing Escherichia coli strains collected in 2015-17. 9Although the sole presence of bla NDM confers sufficiently high-level carbapenem resistance to bacteria, there have been many previous reports on isolates co-carrying bla NDM and bla OXA-48 -like genes. 10Most harbour these two genes on two separate plasmids, whereas only a few co-harbour them on the same plasmid, such as the Acinetobacter plasmid GR59. 11Here, we report an IncX3 plasmid co-harbouring bla NDM-5 and bla OXA-181 and explore the potential advantage of carrying both of these genes through comparative phenotypic analysis with typical endemic IncX3 plasmids harbouring only one of the two genes.

Bacterial isolates and antimicrobial susceptibility testing
An E. coli isolate, JBEHAAB-19-0176, was isolated during the Japan Antimicrobial Resistant Bacterial Surveillance, focusing on Gram-negative bacteria (JARBS-GNR) that had been conducted during 2019-20, which also focused on nosocomial Enterobacterales isolates with reduced carbapenem susceptibility and/or resistance to third-generation cephalosporins. 12The current study was approved by the International Review Board of the National Institute of Infectious Diseases (approval number: 1553).The isolate was recovered from the faeces of an outpatient at the University of Ryukyus Hospital (Okinawa, Japan).Antimicrobial susceptibility testing was performed using the MicroScan WalkAway Plus system with Neg MIC EN 2J and 3.31E panels (Beckman Coulter, Brea, CA, USA).The MICs of meropenem, imipenem, ampicillin, piperacillin and cefpodoxime were further determined using a broth microdilution method, according to the CLSI guidelines.

Transformation and conjugation
Plasmid DNA was introduced into E. coli HST08 cells (Takara Bio, Shiga, Japan) via electroporation using a Gene Pulser Xcell (Bio-Rad, Hercules, CA, USA).The size of the plasmids in the transformants and the presence of bla NDM-5 and bla OXA-181 on them were confirmed through S1 nuclease digestion of whole genomic DNA, followed by PFGE and Southern hybridization, as described previously. 5The conjugation assay was conducted as previously described, with slight modifications, 5 using rifampicinresistant E. coli ML4909 18 as the recipient.Transformants carrying IncX3 plasmids were mixed with recipient cells in a 1:10 ratio and incubated on a nitrocellulose membrane filter at 37°C for 2 h.The bacterial mixture was then suspended in brain heart infusion (BHI) broth and plated onto BHI agar containing meropenem (0.125 mg/L) and rifampicin (100 mg/L).The presence of bla NDM-5 and/or bla OXA-181 in the colonies was confirmed using colony-direct PCR.The conjugation frequency was calculated by dividing the number of cfu of the transconjugants by the number of cfu of the donor and transconjugants.
Competition assays were conducted as previously described, 19 with slight modifications.Bacterial suspensions of the above two transformants were adjusted to OD 600 = 0.5 and mixed at a 1:1 ratio.The mixture was then diluted 100-fold in LB broth with or without piperacillin (128 mg/L), cefpodoxime (64 mg/L) or meropenem (16 mg/L) and incubated at 37°C with shaking.The number of cells in each culture was evaluated at 24 h timepoints by spreading serially diluted cells and then culturing them on two types of BHI agar plates.One plate contained 0.25 mg/L meropenem to count the number of the two transformants, and the other plate contained 0.25 mg/L meropenem and 0.125 mg/L levofloxacin to count the number of pJBEHAAB-19-0176_NDM-OXA transformants carrying the quinolone resistance gene qnrS1.The competition indices were calculated by dividing the number of pJBEHAAB-19-0176_NDM-OXA transformants by the total number of transformants.

Plasmid construction and induction of bla OXA-181 expression
The bla OXA-181 gene was cloned into the arabinose-inducible expression vector, pBAD18-cm, 20 as described previously. 21The pBAD18-cm and the one harbouring bla OXA-181 were introduced into E. coli HST08 carrying the pJBBDAGF-19-0019_NDM-5 plasmid via electroporation.Growth and competition assays were performed as described above in the presence of 0.02% arabinose and 30 mg/L chloramphenicol to induce bla OXA-181 expression and to maintain the introduced plasmids, respectively.For the competition assay, the ratio of E. coli cells with and without bla OXA-181 was determined using PCR.Each culture was spread onto BHI agar plates containing 0.25 mg/L meropenem and 30 mg/L chloramphenicol.At least 20 colonies per condition were subjected to colony-direct PCR using a primer pair targeting the upstream and downstream regions of the pBAD18-cm cloning site (5′-GATTAGCGGATCCTACCTGAC-3′ and 5′-CTTCTCTCATCCGCCAAAAC-3′).Competition indices were calculated by dividing the number of pJBBDAGF-19-0019_NDM-5 transformants positive for a longer insert (i.e.bla OXA-181 ) by the total number of transformants.

Nucleotide sequence accession numbers
The nucleotide sequence of strain JBEHAAB-19-0176 was deposited in DDBJ/ENA/GenBank under the BioSample accession number: SAMD00502403.

Results and discussion
An isolate co-carrying bla NDM-5 and bla OXA-181 , JBEHAAB-19-0176, was obtained from the faeces of an outpatient who had returned from a trip to Bangladesh and was diagnosed with Zuo et al.Campylobacter enteritis.These carbapenemase genes are the most common and the third most common carbapenemase genes encountered in a hospital surveillance in Bangladesh, respectively; 22 however, both genes have rarely been detected in Japan. 12Therefore, it is likely that the isolate was imported from Bangladesh.This strain belonged to ST648, a globally disseminated carbapenem-resistant clone and the first reported isolate with NDM-5 in the UK in 2011. 23Complete sequences showed that the isolates had a chromosome ( 5 1.The strain harboured resistance genes against aminoglycosides (aadA2), sulphonamides (sul1) and trimethoprim (dfrA12) on the chromosome.The quinolone resistance gene qnrS1 was also located on the IncX3 plasmid, together with bla NDM-5 and bla OXA-181 , whereas the macrolide resistance gene mph(A) was present on both the chromosome and IncFIB plasmids.Additionally, bla CMY-141 was located on an IncI (gamma) plasmid.
Dual carriage of carbapenemase genes can contribute to high carbapenem resistance compared with that of organisms with single carriage. 27We generated the E. coli HST08 transformants carrying one of three IncX3 plasmids to investigate the phenotypic differences and potential benefits of co-carrying bla NDM-5 and  Zuo et al.IncX3 plasmid co-harbouring bla NDM-5 and bla OXA-181 bla OXA-181 compared with when either gene is carried alone.Antimicrobial susceptibility testing via a broth microdilution method showed that for the pJBEHAAB-19-0176_NDM-OXA transformants, the carbapenem MICs were identical to those of the pJBBDAGF-19-0019_NDM-5 transformants (Table 2).Interestingly, the MICs of ampicillin, piperacillin and cefpodoxime were 2-fold higher in the pJBEHAAB-19-0176_NDM-OXA transformants than in the pJBBDAGF-19-0019_NDM-5 transformants, indicating that the additional carriage of bla OXA-181 could increase the MICs of these antibiotics.Although pJBCDAAC-19-0068_OXA-181 showed 16 times lower carbapenem MICs than those of the other two transformants, it showed a >16 times higher MIC than that of the host (E. coli HST08).We further investigated the growth rates, i.e. the time to reach the exponential phase, of the two transformants through measuring changes in the OD 600 over time in the presence of different antibiotics.Notably, the growth rate of the pJBEHAAB-19-0176_NDM-OXA transformant was higher than that of the pJBBDAGF-19-0019_NDM-5 transformant in the presence of piperacillin and cefpodoxime (Figure 2a and b).In contrast, both transformants showed comparable growth rates in the presence of meropenem and other β-lactams tested (Figure 2c The transformant carrying a plasmid co-harbouring bla NDM-5 and bla OXA-181 outcompetes one carrying a plasmid with only bla NDM-5 in the presence of piperacillin and cefpodoxime.Bacterial growth curves of pJBEHAAB-19-0176_NDM-OXA (HST::pJBEHAAB-19-0176_NDM-OXA, green) and pJBBDAGF-19-0019_NDM (HST::pJBBDAGF-19-0019_NDM-5, blue) transformants.OD 600 was monitored in the presence of 64 mg/L piperacillin (a), 64 mg/L cefpodoxime (b) or 128 mg/L meropenem (c).The experiment was repeated three times.The pJBEHAAB-19-0176_NDM-OXA and pJBBDAGF-19-0019_NDM-5 transformants were mixed and cultured in the presence or absence of 128 mg/L piperacillin, 64 mg/L cefpodoxime and 16 mg/L meropenem.The bacterial counts were measured 24 h after inoculation, and the abundance ratio of pJBEHAAB-19-0176_NDM-OXA transformants calculated (d).The experiment was repeated eight times.Error bars represent standard deviations.Asterisks show significant differences (Steel test, P < 0.05).
IncX3 plasmid co-harbouring bla NDM-5 and bla OXA-181 and Figure S1, available as Supplementary data at JAC-AMR Online).The pJBEHAAB-19-0176_NDM-OXA transformants reached OD 600 = 0.4 at 7.6 and 7.0 h after being inoculated in the presence of piperacillin and cefpodoxime, respectively.In contrast, the pJBBDAGF-19-0019_NDM-5 transformant took longer to reach OD 600 = 0.4 (11.6 and 21.0 h, respectively) under the same conditions.These results suggest that the bla NDM-5 / bla OXA-181 co-carrier had a growth advantage in the presence of these antibiotics, particularly cefpodoxime, compared with that of the bla NDM-5 single carrier.We tested this using a competition assay in which pJBEHAAB-19-0176_NDM-OXA and pJBBDAGF-19-0019_NDM-5 transformants were mixed and incubated in the presence of the above antibiotics.The bacteria were counted 24 h after inoculation (Figure 2d), and results showed that the growth of these transformants was almost comparable in the absence of any drugs and in the presence of meropenem.In contrast, the pJBEHAAB-19-0176_NDM-OXA transformant outcompeted the pJBBDAGF-19-0019_NDM-5 transformant in the presence of piperacillin and cefpodoxime, suggesting that the additional carriage of bla OXA-181 confers a competitive advantage over bla NDM-5 single carriers under these conditions.
To test this notion further, we compared the growth rate of pJBBDAGF-19-0019_NDM-5 transformants carrying an arabinose-inducible expression vector with (pBAD-OXA-181) or without (pBAD) bla OXA-181 .When the experiment was performed with piperacillin and cefpodoxime, the growth rate of the transformant with pBAD-OXA-181 was faster than that with pBAD in the presence of arabinose (Figure 3a and b).Similar differences were not observed in the absence of arabinose (Figure 3d and e) and in the condition when meropenem was used instead of piperacillin/cefpodoxime (Figure 3c and f).In addition, the pJBBDAGF-19-0019_NDM-5 transformant carrying pBAD-OXA-181 outcompeted the one carrying pBAD in the presence of arabinose and piperacillin or cefpodoxime, but not meropenem (Figure 3g).Thus, the additional expression of bla OXA-181 was sufficient to confer a growth advantage in the presence of piperacillin and cefpodoxime.OXA-48 efficiently hydrolyses piperacillin, and OXA-181 shows similar substrate specificity. 28,29herefore, it is probable that OXA-181 can hydrolyse piperacillin, and the additional presence of its encoding gene provides additional piperacillin hydrolytic activity to the bacteria.In contrast, it is unclear why additional carriage of bla OXA-181 provides a growth advantage in the presence of cefpodoxime because a subset of OXA-48 enzymes, including OXA-181, is incapable of hydrolysing extended-spectrum cephalosporins. 6This issue should be addressed in future studies.
1][32][33] It was also observed that isolates co-carrying the two carbapenemase genes, bla NDM-1 and bla OXA-232 or bla OXA-181 , emerged in three different STs of Klebsiella pneumoniae during a 4 year hospital surveillance, with a concomitant decrease in bla NDM-1 -carrying isolates. 34The present study provides a clue to understanding the changing epidemiology of CPE.Piperacillin and cefpodoxime could be selective agents for the emergence of isolates co-carrying bla NDM and bla OXA-48 -like genes.Meanwhile, it is conceivable that there are other advantages conferred by double carriage of these genes, which were not explored in this study.In addition, it should be noted that the changing epidemiology can result from many other factors, including characteristics of the host bacteria and plasmids carrying these genes, carriage of other antimicrobial resistance genes, and antimicrobial use.
In conclusion, we identified an IncX3 plasmid co-carrying dual carbapenemase genes in a national surveillance study using short-and long-read WGS.The plasmid was formed via the convergence of two highly disseminated plasmids in an endemic region and then imported to Japan.Due to the global spread of such problematic antimicrobial-resistant plasmids, continued surveillance and efforts to limit their further spread are warranted.

Figure 1 .
Figure 1.Genetic features of pJBEHAAB-19-0176_NDM-OXA and proposed models for its formation.(a) Linear comparison of the pJBEHAAB-19-0176_NDM-OXA plasmid with two other IncX3 plasmids harbouring bla NDM-5 or bla OXA-181 .The coding sequences are represented by boxed arrows, and homologous regions (>99% identity) highlighted in grey shading.(b) Schematic diagram of pJBEHAAB-19-0176_NDM-OXA formation.The red line denotes an IncX3 plasmid with bla NDM-5 , and the blue one denotes that with bla OXA-181 .The red and orange arrows indicate IS3000, and the light blue arrows indicate IS26.Flags represent 8 bp flanking sequences of IS26.Wavy arrows show the recombination event.

Table 2 .
Antimicrobial susceptibility patterns of bla